JPH02501108A - Production of human parathyroid hormone by microorganisms - Google Patents
Production of human parathyroid hormone by microorganismsInfo
- Publication number
- JPH02501108A JPH02501108A JP62506628A JP50662887A JPH02501108A JP H02501108 A JPH02501108 A JP H02501108A JP 62506628 A JP62506628 A JP 62506628A JP 50662887 A JP50662887 A JP 50662887A JP H02501108 A JPH02501108 A JP H02501108A
- Authority
- JP
- Japan
- Prior art keywords
- plasmid
- yeast
- microorganism
- dna
- parathyroid hormone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
(57)【要約】本公報は電子出願前の出願データであるため要約のデータは記録されません。 (57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】 微生物によるヒト副甲状腺ホルモンの生産本発明はプレプロヒト副甲状腺ホルモ ンをコードするＤＮＡを持つ、遺伝子工学的操作を加えた微生物に関するもので ある。[Detailed description of the invention] Production of human parathyroid hormone by microorganisms The present invention relates to the production of pre-prohuman parathyroid hormone. It concerns microorganisms that have been genetically engineered and have DNA that codes for be.
哺乳類細胞によって正常に合成される数々の蛋白質やペプチドは、医学、農業、 工業上有用であることが証明されてきた。これらの蛋白質やペプチドは異なる分 子サイズでもあり、例えば酵素や構造蛋白質、成長因子、ホルモンなどのように いくつかの異なる機能を有している。Numerous proteins and peptides normally synthesized by mammalian cells are used in medicine, agriculture, It has been proven to be industrially useful. These proteins and peptides have different It is also the child size, such as enzymes, structural proteins, growth factors, hormones, etc. It has several different functions.
本質的に、蛋白質、ペプチド共に、アミノ酸の一次配列より構成されており、２ 次構造、３次構造を形成して生物学的活性を発現する。Essentially, both proteins and peptides are composed of a primary sequence of amino acids. It forms secondary and tertiary structures to express biological activity.
ヒト副甲状腺ホルモンは比較的小分子量であり、アミノ酸を順次付加することに よって、化学的にこのペプチドを合成する事が可能である。Human parathyroid hormone has a relatively small molecular weight, and it is possible to add amino acids sequentially. Therefore, it is possible to chemically synthesize this peptide.
故に副甲状腺ホルモンは商業的に利用可能ではあるが、ごく少量で高価である。Therefore, although parathyroid hormone is commercially available, it is available in very small quantities and is expensive.
結果的に多くの潜在的、医学、農業、工業面の応用に供するのに適当な価格の利 用可能なヒト副甲状腺ホルモンは存在しない。As a result, it has many potential medical, agricultural, and industrial applications at a reasonable price. There is no human parathyroid hormone available.
過去１０年間、組み換えＤＮＡを用いた微生物学的技術は異種のペプチドの生産 に微生物を利用する事を可能にしてきた。微生物は急速かつ、大量の成長が可能 であり、細菌のペプチドと同様の方法で、外来性の産生物を合成出来る。この分 子生物学的アプローチの有効性と可能性は既に医学或いは他の使用目的に今や多 用可能な数々のヒト蛋白質の微生物による生産によって証明されてきた。Over the past decade, microbiological techniques using recombinant DNA have been used to produce heterologous peptides. It has made it possible to use microorganisms in Microorganisms can grow rapidly and in large quantities , and foreign products can be synthesized in a manner similar to bacterial peptides. this minute The effectiveness and potential of biobiological approaches are now widely recognized for medical and other uses. Microbial production of a number of usable human proteins has been demonstrated.
副甲状腺ホルモン（ＰＴＨ）は哺乳類のカルシウム代謝の最も重要なレギュレー ターの１つであり、ヒトや動物のミルク熱急性低カルシウム症の他に、病理的血 中カルシウムレベル変化といった、いくつかの病気にも関連している。それ故に このホルモンは、診断キットの一部としても重要であり、ヒトや家畜の医療にお いても治療に潜在的可能性を有している。Parathyroid hormone (PTH) is the most important regulator of calcium metabolism in mammals. In addition to milk fever acute hypocalcemia in humans and animals, it also causes pathological blood loss. It is also associated with several diseases, such as changes in intermediate calcium levels. Therefore This hormone is also important as part of diagnostic kits and is used in human and livestock medicine. However, it has therapeutic potential.
ヒトプレプロ副甲状腺ホルモンＤＮへの最初の合成はヘントリ（Ｈｅｎｄｒｙ） Ｇ、Ｎ、Ｋｒｏｎｅｂｅｒｇ、）１．Ｍ、、Ｐｏｔｔｓ、Ｊｒ、Ｊ、Ｔ、Ｒｉｃ ｈ Ａらによって報告された。（７８Ｐｒｏｃ、Ｎａｔｌ Ａｃａｄ、Ｓｃｉ、 ７３６５−７３６９．１９８１） 。The first synthesis of human preproparathyroid hormone DN was by Hendry. G., N. Kroneberg, ) 1. M., Potts, Jr., J., T., Ric. h Reported by A et al. (78Proc, Natl Acad, Sci, 7365-7369.1981).
またヘンドリーらによってＰＴＨ，ｎ＋ＲＮＡ配列の相補ＤＮＡが合成され、２ 本鎖に合成された（前出）、このｃＤＮＡは、ｐＢＲ３２２ＤＮＡにクローニン グされ、大腸菌（Ｅ、ｃｏｌｉ、） １７７６にトランスフェクトされた。選択 された抗生物質に耐性のコロニーのうち、２００コロニー中２３コロニーが、特 異的ヒトＰＴＨｃＤＮＡの挿入を持っていることが同定された。しかしながら２ ３個のヒトＰＴ）ｌコロニーのうち、全長にわたって挿入されているものはなか った（ヘンドリーら、前出）、後にプレイエル（Ｂｒｅｙｅｌ）、Ｅ、］’１ｏ ｒｅｌｌｅ、Ｇ、 Ａｕｆ’ｍｔｏｌｋ、Ｂ。Furthermore, complementary DNA of the PTH,n+RNA sequence was synthesized by Hendry et al. This cDNA, synthesized as a full-strand (see above), was cloned into pBR322DNA. and transfected into Escherichia coli (E. coli) 1776. choice Of the colonies resistant to the antibiotics tested, 23 out of 200 were It was identified that it had an insertion of a heterologous human PTH cDNA. However, 2 Of the 3 human PT) colonies, few had full-length insertions. (Hendry et al., supra), later Breyel, E.]'1o relle, G, Auf'mtolk, B.
Ｆｒａｎｋ、Ｒ，Ｂｌｏｃｋｅｒ Ｈ，Ｍａｙｅｒ、）１．らは、シャロン４Ａ フアージで構成された胎児肝、ジェノミックＤＮＡライブラリーにヒ） ＰＴ） ｌ遺伝子があることを報告した（第３回欧州バイオテクノロジー会議、１９８４ 、 ９月１０ １４． ｖｏｌ、３．３６３ ３６９）。ＰＴＨ遺伝子の制限酵 素断片が再クローン化され、大腸菌（Ｅ、ｃｏｌｉ、）にトランスフェクトされ た。Frank, R, Blocker H, Mayer) 1. et al. Sharon 4A Fetal liver composed of Phage, genomic DNA library (PT) reported that there is a l gene (3rd European Biotechnology Conference, 1984 , September 10, 14. vol, 3.363 369). Restriction enzyme of PTH gene The elementary fragment was recloned and transfected into E. coli. Ta.
しかし、プレイエルらの研究により（前出） 、Ｅ、ｃｏｌｉはヒトＰＴ）ｌを 分解することがわかった。故に、これまでのところ、無障害のヒト副甲状腺ホル モンを安定に生産する微生物は報告されてきていない。更に、副甲状腺ホルモン は酵母からも単離されてきていない。However, according to research by Pleyel et al. (cited above), E. coli has a human PT) It was found that it can be decomposed. Therefore, so far, unimpaired human parathyroid hormone There have been no reports of microorganisms that stably produce Mon. Additionally, parathyroid hormone has not been isolated from yeast either.
したがって、ヒトプレプロ副甲状腺ホルモン（）ＩＰＴ）ｌ）をコードするＤＮ Ａを持つ大腸菌（Ｅ、ｃｏｌｉ）挿入用のプラスミドの開発が本発明の目的であ る。また、ヒトプレプロ副甲状腺ホルモンをコードするＤＮＡを持つ大腸菌を遺 伝子工学的に作成する事が本発明のもう一つの目的である。Therefore, the DN encoding human preproparathyroid hormone ()IPT)l) The purpose of the present invention is to develop a plasmid for insertion into Escherichia coli (E. coli) carrying A. Ru. In addition, E. coli containing DNA encoding human preproparathyroid hormone was found. Genetic engineering is another object of the present invention.
更に、本発明の目的は、酵母挿入用の副甲状腺ホルモンをコードするＤＮＡを持 つプラスミドの開発である。また、ヒト副甲状腺ホルモンを含めて、副甲状腺ホ ルモンをコードするＤＮＡを持つ、形質転換酵母を作成し、その形質転換酵母か ら、副甲状腺ホルモンを得ることが、本発明の目的である。Furthermore, it is an object of the present invention to carry the DNA encoding parathyroid hormone for yeast insertion. This is the development of two plasmids. In addition, parathyroid hormone, including human parathyroid hormone, Create a transformed yeast that has DNA encoding rumon, and then It is an object of the present invention to obtain parathyroid hormone.
本発明の他の目的や、利用法は、後述されるところによって明らかとなるであろ う。Other objects and uses of the present invention will become apparent from the description below. cormorant.
前述の目的を達成する為に、本発明によってヒトプレプロ副甲状腺ホルモンをコ ードするＤＮＡを持つ大腸菌挿入用の新しいプラスミドが作成された。このプラ スミドは大腸菌に挿入されると大腸菌を形質転換させ、マルチコピーのプラスミ ド、つまりヒトプレプロ副甲状腺ホルモンのｃＤＮＡを複製させるものとして機 能する０本発明による大腸菌挿入用のプラスミドと形質転換大腸菌は、例えば、 ヘンドリーらによって報告されているように、本発明のプラスミドは、プレプロ 副甲状腺ホルモンをコードするＤＮＡの５′未満に２重の開始コドンを含んでい る点で、今までの人ロブラスミドや微生物と区別できる。この２重の開始コドン の存在により、このｃＤＮＡを持つプラスミドで微生物を形質転換させた時、プ レプロ副甲状腺ホルモンを従来技術の形質転換微生物よりも高効率、高収量で生 産するような生産微生物が得られたのであろう。In order to achieve the aforementioned objectives, human preproparathyroid hormone is co-coated according to the present invention. A new plasmid for E. coli insertion was created with the DNA encoding the gene. This plastic When inserted into E. coli, the sumid transforms the bacterium and creates a multicopy plasmid. In other words, it functions as a cDNA for human preproparathyroid hormone. The plasmid for insertion into E. coli and the transformed E. coli according to the present invention are, for example, As reported by Hendry et al., the plasmid of the present invention The DNA encoding parathyroid hormone contains a double start codon in the 5′ region. It can be distinguished from conventional human loblastmids and microorganisms in that This double start codon Due to the presence of this cDNA, when microorganisms are transformed with a plasmid containing this cDNA, Produces reproparathyroid hormone with higher efficiency and yield than conventional transformed microorganisms. It is likely that the microorganisms that produced the product were obtained.
更に、本発明によって副甲状腺ホルモンをコードするＤＮＡを持った酵母挿入用 のプラスミドが作成された。より具体的にはこのプラスミドは前述の、大腸菌へ の挿入用のプラスミドをクローン化することによって開発された。最終的には、 本発明により、前述の酵母への挿入用プラスミドによって副甲状腺ホルモンを生 産分泌するような形質転換酵母が得られた。即ち、本発明は、酵母培地から、副 甲状腺ホルモンを単離できるような方法を開発したものである。Furthermore, according to the present invention, a yeast insert containing DNA encoding parathyroid hormone can be used. A plasmid was created. More specifically, this plasmid is transferred to E. coli as described above. was developed by cloning a plasmid for the insertion of eventually, According to the present invention, parathyroid hormone is produced by the aforementioned plasmid for insertion into yeast. A transformed yeast capable of producing and secreting was obtained. That is, the present invention enables the production of supplements from yeast medium. A method was developed to isolate thyroid hormone.
より具体的には、この形質転換酵母は、サツカロマイセス・セレビシアエ（Ｓａ ｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）である。また、副甲状腺ホル モンは、ヒト副甲状腺ホルモンである。ブタペスト条約の規定により、ｐｓｓＨ ＰＴＨ−１０とその形質転換大腸菌、ｐＳＳαＬＸ５−）ＩＰＴＨＩとその形質 転換サツカロマイセス・セレビシアエ（Ｓａｃｃｈａｒｏｓ＋ｙｃｅｓ ｃｅｒ ｅｖｉｓｉａｅ）は、１９８６年９月２９日、メリーランド州のアメリカン・タ イプ・カルチャー・コレクション（Ａ＋＊ｅｒｉｃａｎ Ｔｙｐｅ Ｃｕ１ｔｕ ｒｅ Ｃｏ１１ｅｃｔｉｏｎ）に供託された。これらのサンプルは、以下の寄託 番号を付与されている。More specifically, this transformed yeast is Satucharomyces cerevisiae (Sa ccharomyces cerevisiae). Also, parathyroid hormone Mon is human parathyroid hormone. According to the provisions of the Budapest Treaty, pssH PTH-10 and its transformed E. coli, pSSαLX5-)IPTHI and its characteristics Conversion Saccharomyces cerevisiae (Saccharos+yces cer evisiae) was born on September 29, 1986, in the American state of Maryland. Ip Culture Collection (A++ erican Type Cu1tu re Co11ection). These samples have been deposited below. is assigned a number.
ｐＳＳ）ＩＰＴ）ｌ−１０を持つ、形質転換大腸菌 ＡＴＣＣ６７２２３ｐｓｓ ＨＰＴＨ−１０ＡＴＣＣ４０２６７ｐＳＳαＬＸＳ−ＨＰＴＨ１を持つ形質転換 サツカロマイセス・セレビシアエ ＡＴＣＣ２０８２１ｐｓｓαＬＸ５−ＨＰＴ ＨＩ ＡＴＣＣ４０２６６図１にヒトプレプロ副甲状腺ホルモンをコードするＤ ＮＡ塩基配列の可能な全てのバリエーションを示しである。Transformed E. coli ATCC67223pss carrying pSS)IPT)l-10 Transformation with HPTH-10ATCC40267pSSαLXS-HPTH1 Satucharomyces cerevisiae ATCC20821pssαLX5-HPT HI ATCC40266 Figure 1 shows D encoding human preproparathyroid hormone All possible variations of the NA base sequence are shown.
図２にクローンｐＳＳ）ＩＰＴ）Ｉ−１０のヒトプレプロ副甲状腺ホルモンＤＮ Ａに特異的なコード配列を示しである。Figure 2 shows human preproparathyroid hormone DN of clone pSS)IPT)I-10. The coding sequence specific to A is shown.
図３に本発明のプラスミドに存在すると考えられるヒトプレプロ副甲状腺ホルモ ンをコードし、５′末端に２重のスタートコドンを持つ、可能な全てのＤＮＡ配 列をその近接領域と共に示している。Figure 3 shows the human preproparathyroid hormone that is thought to be present in the plasmid of the present invention. All possible DNA sequences encoding the A column is shown along with its adjacent area.
図４はクローンｐＳＳ）ＩＰＴ）ｌ−１０の特異的ヒトプレプロ副甲状腺ホルモ ンＤＮＡ配列をその近接配列と共に示しである。Figure 4 shows the specific human preproparathyroid hormone of clone pSS)IPT)l-10. The DNA sequence is shown together with its neighboring sequences.
図５はクローンｐＳＳ）ＩＰＴＨ−１０のＤＮＡ配列にコードされる、ヒトプレ プロ副甲状腺ホルモンの実際のアミノ酸配列を示している。Figure 5 shows the human protein sequence encoded by the DNA sequence of clone pSS) IPTH-10. Showing the actual amino acid sequence of pro-parathyroid hormone.
図６は組み換えプラスミドｐＳＳＨＰＴＨ−１０の構成を示している。Figure 6 shows the structure of recombinant plasmid pSSHPTH-10.
図７はｐＡＬＸ ４の制限地図を示している。Figure 7 shows the restriction map of pALX4.
図８はｐＬ４とｐＭＦα１−１からのｐαＬＸ−５の構成を示している。Figure 8 shows the composition of pαLX-5 from pL4 and pMFα1-1.
図９はｐＳＳαＬＸ５−ＨＰＴＨ１の概略図を示している。Figure 9 shows a schematic diagram of pSSαLX5-HPTH1.
図１０は肝α１−ＨＰＴＨの融合遺伝子の配列と、肝ＴＨをコードするＤＮＡの 可能な全ての組み合わせを示している。Figure 10 shows the sequence of the liver α1-HPTH fusion gene and the DNA encoding liver TH. Showing all possible combinations.
図１１は肝α１−ＨＰＴ）Ｉの融合遺伝子の配列を示している。FIG. 11 shows the sequence of the liver α1-HPT)I fusion gene.
図１２は酵母が生産、分泌し、酵母培地から回収されたヒト副甲状腺ホルモンの 電気泳動プレートを示している。Figure 12 shows human parathyroid hormone produced and secreted by yeast and recovered from yeast culture medium. An electrophoresis plate is shown.
以上に示したように本発明は、ヒトプレプロ副甲状腺ホルモンをコードするＤＮ Ａを含んだ大腸菌への挿入用プラスミドについてのものである。また本発明は作 成された形質転換大腸菌に関する。As shown above, the present invention provides a DN encoding human preproparathyroid hormone. This is a plasmid for insertion into E. coli containing A. Moreover, the present invention The present invention relates to the transformed E. coli.
更に本発明は、大腸直への挿入用プラスミドに由来する、副甲状腺ホルモンをコ ードするＤＮＡを持つ酵母への挿入用プラスミドについてのものである。最終的 には本発明は、副甲状腺ホルモンが回収されると考えられる酵母についてのもの である。Furthermore, the present invention provides a method for co-producing parathyroid hormone derived from a plasmid for insertion into the rectum. This is a plasmid for insertion into yeast that contains the DNA coded for. Final The present invention relates to yeast from which parathyroid hormone is thought to be recovered. It is.
更に本発明によりこのプラスミドと形質転換微生物の単離と作成の方法がもたら される。外科的操作後直ちに回収されたヒト副甲状腺腫からポリＡ選択のｌ’ｌ ＮＡが単離された。ポリＡ ＲＮＡを庶糖濃度勾配超遠心によって適正な大きさ の１ｌＲＮＡに濃縮した。Furthermore, the present invention provides methods for isolating and producing this plasmid and transformed microorganisms. be done. PolyA-selected l’l from human parathyroid tumors recovered immediately after surgical manipulation. NA was isolated. Poly A RNA is reduced to an appropriate size by sucrose concentration gradient ultracentrifugation. of RNA.
このサイズに分画されたポリＡ ＲＮＡからインビトロで正常な分子量の副甲状 腺ホルモンを翻訳しこれを、抗副甲状腺腫抗血清による免疫沈降後に、ＳＯＳ電 気泳動によって判定した。ヒト、ＰＴＨ特異的ｍＲＮＡを鋳型として用いオリゴ ｄ （Ｔ） １８マーをプライマーとして鳥筋原細胞腫ウィルス逆転写酵素を用 いて相補ＤＮＡを合成した。 ＲＮＡの鋳型をアルカリ加水分解によって除去し た後に、大腸菌ＤＮＡポリメラーゼ■のフレノウフラグメント存在下で精製した 第１のＤＮＡ鎖をインキュベートすることによって第２の相補鎖ＤＮＡを合成し た。二本鎖の相補ＤＮＡの末端をアスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌ ｌｕｓ ｏｒｙｚａｅ）の−末鎖特異的エンドヌクレアーゼ５１の作用により平 滑末端にし、５ｏｏｂｐ以上の長さの相補ＤＮＡをニュートラル庶糖濃度勾配遠 心によって単離した。約２０塩基長のｄ （Ｃ）テイルをｃＤＮＡの３′末端に 酵素によって付加した。この修飾された相補ＤＮＡを制限酵素ＰｓｔＩで切断し オリゴｄ （Ｇ）テイルを付加したベクターｐＢＲ３２２に付加した。作成され た組み換えプラスミドＤＮＡを大腸菌に１２ ＢＪ５１０３株に形質転換させた 。ポジティブな形質転換株は、それぞれ、ホルモンｍＲＮＡ配列の３′末端の非 コード領域と、Ｎ本のコード領域をカバーする２種類の合成デオキシオリゴヌク レオチドを用いたコロニーハイブリダイゼーションによって検索した。６６個の クローンのうち６つが両方のプローブにポジティブであり、これらを制限酵素地 図による詳細な解析に供したところ、スタートコドンと３′末端のストップコド ンのＸｂａ １部位の近接領域において、サイズがいくつか異なることを除いて は、全て同一であった。そのうちの１つのクローンであるｐｓｓＨＰＴＨ４０の ＤＮＡ配列を解析したところｐＢＲ３２２のＰｓｔ１部位に４３２塩基長のヒト 副甲状腺ホルモンの相補ＤＮＡが挿入されている事が判明した。この全ｃＤＮＡ 配列は、スタートコドンの前に５ｂｐの欠失があった事を除いては、ヘンドリー ら（前述）によって報告された配列と同一であった。Poly A RNA fractionated to this size was used in vitro to obtain normal molecular weight parathyroid tissue. The glandular hormones were translated and subjected to SOS electrolysis after immunoprecipitation with anti-parathyroidoma antiserum. Determined by pneumophoresis. Oligos using human, PTH-specific mRNA as a template d (T) Using avian myoblastoma virus reverse transcriptase using 18-mer as a primer Complementary DNA was synthesized. Remove the RNA template by alkaline hydrolysis After that, it was purified in the presence of the Flenow fragment of E. coli DNA polymerase. Synthesize a second complementary strand DNA by incubating the first DNA strand Ta. The ends of the double-stranded complementary DNA were attached to Aspergillus oryzae. by the action of the -terminal strand-specific endonuclease 51 of Complementary DNA with a length of 5 oobp or more was made into a blunt end and was passed through a neutral sucrose concentration gradient. Isolated by heart. Add a d(C) tail of approximately 20 bases to the 3' end of the cDNA. Added by enzyme. This modified complementary DNA was cut with the restriction enzyme PstI. It was added to vector pBR322 with an oligo d(G) tail added. created The recombinant plasmid DNA was transformed into E. coli strain 12 BJ5103. . Positive transformants each contain a non-3' end of the hormone mRNA sequence. coding region and two types of synthetic deoxyoligonucs covering N coding regions. The search was performed by colony hybridization using leotide. 66 pieces Six of the clones were positive for both probes, and these were When subjected to detailed diagrammatic analysis, it was found that the start codon and the stop codon at the 3' end Except for some differences in size in the vicinity of the Xba 1 site of were all the same. One of the clones, pssHPTH40, Analysis of the DNA sequence revealed that a 432 base long human DNA was found in the Pst1 site of pBR322. It was discovered that complementary DNA for parathyroid hormone had been inserted. This entire cDNA The sequence is Hendry except for a 5 bp deletion before the start codon. (supra).
図２は、ｐｓｓＨＰＴＨ−１０のヒトプレプロ副甲状腺ホルモンのＤＮＡ配列を 示したものである。５′の二重スタートコドンを除いては、ヒト副甲状腺ホルモ ンのＤＮＡ配列の可能な全てのバリエーションを示す図１と一致するであろう。Figure 2 shows the DNA sequence of human preproparathyroid hormone of pssHPTH-10. This is what is shown. Except for the 5' double start codon, human parathyroid hormone Figure 1 shows all possible variations of the DNA sequence of the DNA sequence.
図３は本発明によって得られたクローンのＤＮＡ配列をその近接領域の配列と共 に示しである。より具体的には、ヒト副甲状腺ホルモンをコードする大腸菌への 挿入用プラスミドはｐＳＳＨＰＴＨ−１０であり、そのＤＮＡ配列は近傍の配列 と共に図４に示しである。Figure 3 shows the DNA sequence of the clone obtained by the present invention together with the sequence of its adjacent region. This is shown below. More specifically, to E. coli encoding human parathyroid hormone. The insertion plasmid is pSSHPTH-10, and its DNA sequence is similar to the neighboring sequence. This is shown in FIG.
更に本発明により、酵母への挿入用の、副甲状腺をコードする１１１ＮＡを持つ プラスミドが得られる。この副甲状腺ホルモンはヒト及び動物、例えばブタやウ シの副甲状腺ホルモンでもよい。Furthermore, according to the present invention, we have 111NA encoding the parathyroid gland for insertion into yeast. A plasmid is obtained. This parathyroid hormone is produced in humans and animals, such as pigs and horses. The parathyroid hormone of Shi may also be used.
本発明の酵母挿入用プラスミドは、ヒト又は動物の副甲状腺ホルモンをコードす るＤＮＡを持つプラスミドから再クローン化され得よう、具体的には、酵母への 挿入用プラスミドに、ヒト副甲状腺ホルモンをコードするＤＮＡが含まれている 。以下の例で示すように、ｐｓｓＨＰＴＨ−１０由来のヒ） ）ＩＰＴＨ配列を 再クローン化しサツ力ロマイセスーセレビシアエ（Ｓａｃｃｈａｒｏ＋ｎｙｃｅ ｓ ｃｅｒｅｖｉｓｉａｅ）で発現するように特別に設計されたベクターに挿入 した。The yeast insertion plasmid of the present invention encodes human or animal parathyroid hormone. plasmid containing the DNA, specifically, into yeast. The insertion plasmid contains DNA encoding human parathyroid hormone. . As shown in the example below, the human IPTH sequence derived from pssHPTH-10 is Re-cloned Saccharomyces cerevisiae (Saccharo+nyce) inserted into a specifically designed vector for expression in S. cerevisiae did.
ｐＳＳＨＰＴＨ−１０は２８８ｂｐのＢｇｌ ＩＩ−χｂａＩ断片にあるように 切断した。この断片をｐＵｃ１９中のＢａｍＨＩとＸｂａ １部位間にサブクロ ーニングした。このサブクローンを更にＤｐｎ ｌで切断し生じてきた最も大き な断片を単離した。この断片を次にＳａｌ Ｉで切断した。pSSHPTH-10 is a 288 bp Bgl II-χba I fragment. Amputated. This fragment was subcloned between the BamHI and Xba sites in pUc19. -ning. This subclone was further cut with Dpnl, resulting in the largest A fragment was isolated. This fragment was then cut with SalI.
酵母ＭＡＴα細胞中でＰＴＨを発現、分泌するこのプラスミドｐＳＳαＬＸ−） ＩＰＴＨＩは三段階を経て作成された。This plasmid pSSαLX-) expresses and secretes PTH in yeast MATα cells. IPTHI was developed in three stages.
１、 酵母シャトルベクターｐＬ４ （大腸菌中でもサツカロマイセス・セレビ シアエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）中でも複製する ）の作成。1. Yeast shuttle vector pL4 (E. coli, including Saccharomyces cerevis) It also reproduces in Saccharomyces cerevisiae. ) creation.
２、酵母性フェロモン肝α１遺伝子を含んだＤＮＡ断片のクローニングと、これ の酵母シャトルベクターへの挿入によるｐαＬＸ５ベクターの作成。2. Cloning of a DNA fragment containing the yeast pheromone liver α1 gene and its use Generation of pαLX5 vector by insertion of pαLX5 into yeast shuttle vector.
３、ｐｓｓＨＰＴＨ−１０のＨＰＴＨ遺伝子のコード領域のＤＮＡ断片をｐαＬ Ｘ５のコード領域にＭＦα１遺伝子の前駆体部分と共に挿入することによるｐｓ ｓαＬＸ ５− ＨＰＴＨベクターの作成。3. The DNA fragment of the coding region of the HPTH gene of pssHPTH-10 was converted into pαL ps by inserting the precursor part of the MFα1 gene into the coding region of X5. Creation of sαLX 5-HPTH vector.
シャトルベクターｐＬ４はｐＡＤＨＯ４０より単離したＡＤＨプロモーターを含 む、ＥｃｏＲＩ −Ａｖａ ＩＩ断片をｐＪＤＢ２０７に挿入することによって 作成した。５ｐｈｌ断片を次に欠失させ、プラスミドｐＡＬＸ １を作成した。Shuttle vector pL4 contains the ADH promoter isolated from pADHO40. By inserting the EcoRI-Ava II fragment into pJDB207, Created. The 5phl fragment was then deleted to create plasmid pALX1.
βラクタマーゼ遺伝子中のｐｓｔ１部位を欠失させ、このプラスミドをｐｖａ ＩとＢｇｌ Ｉで部分分解し、ｐＬｌｃのＰｖｕ Ｉ 。The pst1 site in the β-lactamase gene was deleted and this plasmid was transformed into pva Partially decomposed with I and BglI, PvuI of pLlc.
Ｂｇｌ Ｉ断片をライゲーションし、ｐＡＬＸ ２を作成した。さらに、オリゴ ヌクレオチドの挿入後このプラスミドと旧ｎｄＩ［Ｉで分解し再び結合させてｐ ＡＬＸ ４を作成した。The BglI fragment was ligated to create pALX2. In addition, oligo After inserting the nucleotide, this plasmid is digested with old ndI[I and recombined to make p ALX 4 was created.
Ｙ２８８ｃ株より得た酵母の全ＤＮＡをＥｃｏＲＩで切断し１．６〜１．８ｋｂ の断片を単離した。これをＥｃｏＲＩで切断したｐＢＲ３２２に結合し、大腸菌 を形質転換させた。オリゴヌクレオチドハイブリダイゼーションにより肝α−１ の挿入クローンをスクリーニングした。The total yeast DNA obtained from the Y288c strain was cut with EcoRI to 1.6-1.8 kb. A fragment of was isolated. This was ligated to pBR322 cut with EcoRI, and E. coli was transformed. Hepatic α-1 by oligonucleotide hybridization inserted clones were screened.
この結果選択されたＤＮＡを大腸菌の形質転換用に用いた。得られたプラスミド ｐ？ＩＦα１−１をＥｃｏＲＩで切断しフレノウフラグメントで平滑末端にした 後にＢｇｌｌｌで切断した。ＭＦαｌ断片を単離し、（ＢａｍＨＩで切断した後 にフレノウフラグメントで平滑末端にした）ｐＬ５に結合してｐαＬＸ５を作成 した。ｐαＬＸ５にヒ） ＰＴＨｃＤＮＡ断片を挿入する為に、ｐαＬＸ５を旧 ｎｄｌＩｌで切断し、生じた接着末端とこの部位をＤＮＡポリメラーゼ■のフレ ノウフラグメントとｄＮＴＰで平滑末端にした。次にｐαＬχ５を５ａｌｌで切 断し、５ａｉｌで切断した上記のヒトＰＴＨ断片と相補する接着末端を有するＤ ＮＡを作成した。The DNA selected as a result was used for transformation of E. coli. Obtained plasmid p? IFα1-1 was cut with EcoRI and made blunt ended with Frenow fragment. It was then cut with Bglll. The MFαl fragment was isolated (after cutting with BamHI) ligated to pL5 (blunt-ended with Flenow fragment) to create pαLX5. did. In order to insert the PTH cDNA fragment into pαLX5, pαLX5 was Cut with ndlIl, and insert the resulting sticky end and this site into the frame of DNA polymerase ■. The ends were made blunt with Know fragment and dNTP. Next, cut pαLχ5 at 5all. D with cohesive ends complementary to the above human PTH fragment cut with 5ail. Created NA.
５ａｉｌで切断したヒトＰＴ）Ｉ断片を、５ａｉｌで切断したｐαＬＸ５に挿入 した０作成したプラスミドｐＳＳαＬＸ５・ＰＴ）Ｉは図９に示しである。Inserting human PT)I fragment cut with 5ail into pαLX5 cut with 5ail The constructed plasmid pSSαLX5·PT)I is shown in FIG.
ｐｓｓαＬＸ５−ＰＴ）ｌを酵母に挿入し、ヒト副甲状腺ホルモンを生産、分泌 するような形質転換酵母を得た。より具体的には、この形質転換酵母はサツカロ マイセス・セレビシアエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ ）である。pssαLX5-PT)l was inserted into yeast to produce and secrete human parathyroid hormone. A transformed yeast was obtained. More specifically, this transformed yeast Myces cerevisiae (Saccharomyces cerevisiae) ).
図１２に酵母培地より得たヒト副甲状腺ホルモンの電気泳動プレートを示しであ る。Figure 12 shows an electrophoresis plate for human parathyroid hormone obtained from yeast medium. Ru.
しかしながら、ｐＳＳＨＰＴＨ−１０を再クローニングし酵母への挿入用プラス ミドを作成する方法は詳細に示してあり、この方法は本発明を例証するものとし て示しであるが、本発明は以後これに制限されるものではない。この方法は、ヒ ト、動物のＰＴ）ＩをコードするＤＮＡを持つ様々なプラスミドに応用されるだ ろうし、本発明の酵母への挿入用プラスミドが作成出来る。However, pSSHPTH-10 was recloned and inserted into yeast. The method of making the mido is shown in detail and is intended to be illustrative of the invention. However, the present invention is not limited thereto. This method It has been applied to various plasmids containing DNA encoding PT, animal PT)I. plasmid for insertion into yeast according to the present invention.
本発明のプラスミド及び形質転換微生物は以下の例にあるように作成された。The plasmids and transformed microorganisms of the present invention were constructed as in the following examples.
例１ ヒト５ ホルモンのｍＲＮへの −と ＤＮＡ０人本発明の最初の材料として患 者より外科的に得られた副甲状腺腫が使われた。副甲状腺組織を摘出後、直ちに ドライアイス中で冷却しＲＮＡｍ製用に研究室に運ノしだ。この凍結組織は、４ ｈグアニジンイソチオシアネート存在下、超ツラックスホモゲナイザーでホモゲ ナイズし、ガーグイン（Ｇｈｉｒｇ賃ｉｎ Ｊ、Ｍ、）、ビジビラ（Ｐｒｚｙｂ ｙｌａ Ａ、Ｅ、）、マクドナルド（ＭａｃＤｏｎａｌｄ Ｒ，Ｊ、） 、ルタ ー（Ｒｕｔｔｅｒ Ｗ、Ｊ、）らの方法（Ｂｉｏｃｈｅｍｉｓｔｒｙ １８５２ ９４ ５２９９１９７９）に従って段階別エタノール沈殿によってＲＮＡを回収 した。Example 1 Human 5 hormones to mRNA - and DNA 0 human patients as the first material of the present invention. A parathyroid tumor surgically obtained from a patient was used. Immediately after removing the parathyroid tissue It was cooled in dry ice and sent to the laboratory for RNAm production. This frozen tissue is Homogenize using a Super Turrax homogenizer in the presence of h-guanidine isothiocyanate. Ghirg in J, M, Przyb yla A, E,), MacDonald R, J,), Ruta - (Rutter W, J,) et al.'s method (Biochemistry 1852) 94 52991979) by stepwise ethanol precipitation. did.
調製したＲＮＡを、オリゴｄ （Ｔ）セルロース′アフィニティクロマトグラフ ィカラムにかけ、ポリ（Ａ）を持つｍＲＮＡを濃縮した。ポリ（Ａ）を多く含む ＲＮＡを更に１５〜３０％連続庶糖濃度勾配超遠心にかけ、副甲状腺ホルモンｍ ＲＮＡ０サイズのＩ？ＮＡを濃縮した。得られたグラジェントを２５画分に分画 し、３両分毎にインヒドロで翻訳させ、抗ＰＴ）ｌ抗血清による免疫沈降しガウ テビイク（Ｇａｕｔｖｉｋ。The prepared RNA was subjected to oligo d(T)cellulose' affinity chromatography. The mixture was applied to a microcolumn to concentrate mRNA containing poly(A). Contains a lot of poly(A) The RNA was further subjected to 15-30% continuous sucrose gradient ultracentrifugation, and parathyroid hormone m RNA0 size I? NA was concentrated. Fractionate the obtained gradient into 25 fractions Translated with inhydrolyte every 3 minutes and immunoprecipitated with anti-PT)1 antiserum. Gautvik.
Ｋ、Ｍ、） Ｃａｕｔｉｖｉｋ、Ｖ、Ｔ、 Ｈａｌｖｏｒｓｅｎ Ｊ、Ｆ、 ５ ｃａｎｄ、Ｊ、Ｃｌ１ｎ、Ｌａｂ。K, M,) Cautivik, V, T, Halvorsen J, F, 5 cand, J., Cl1n, Lab.
Ｉｎｖｅｓｔ、 ４３５５３−５６９１９８４）とＳＤＳ、ポリアクリルアミド 電気泳動レムリ−（Ｌａｅｍｍｅｌｉ、Ｕ、に、） ２２７ Ｎａｔｕｒｅ ６ ８０１９７０）によってＰＴＨｍＲＮＡ含量を検量した。翻訳可能なＰＴＨｍＲ ＮＡを含む両分のＲＮＡはエタノール沈殿によって回収した。 ＰＴＨ＋＋＋Ｒ ＮＡを多量に含むこのＲＮＡを、ｃＤＮＡ合成の為の鋳型にして、プライマーと してオリゴｄ（Ｔ）１８マーを、反応触媒として鳥筋原細胞腫ウィルス逆転写酵 素を用いた。マニアティス（Ｍａｎｉａｔｉｓ、Ｔ、）Ｆｒｉｔｓｃｈ Ｅ、Ｆ 。Invest, 43553-5691984) and SDS, polyacrylamide Electrophoresis Laemmeli (Laemmeli, U.) 227 Nature 6 PTH mRNA content was calibrated by 801970). Translatable PTHmR Both RNAs containing NA were recovered by ethanol precipitation. PTH＋＋＋R This RNA, which contains a large amount of NA, is used as a template for cDNA synthesis and is combined with primers. The oligo d(T)18mer was used as a reaction catalyst with avian myoblastoma virus reverse transcriptase. The raw material was used. Maniatis (T.) Fritsch E, F .
５ａｎｂｒｏｏｋ、Ｊ、 Ｍｏ１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ｐｐ２３０− ２４３１９８２） ＊第一のＤＮＡ鎖の合成後に、ＲＮＡの鋳型をアルカリ加水 分解によって除去した。第２のｃＤＮＡ鎖は、大腸菌ＤＮＡポリメラーゼＩのフ レノウフラグメント存在下に、精製した第一のｃＤＮＡ鎖をインキュベートして 合成した（マニアナイス前出）。このインビトロ合成二本鎖ｃＤＮＡは、アスペ ルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）の−重鎖特異的 エンドヌクレアーゼＳ１の作用によって平滑末端にした（マニアナイス前出）。5anbrook, J, Molecular Cloning pp230- 2431982) *After synthesis of the first DNA strand, the RNA template is alkaline hydrated. Removed by decomposition. The second cDNA strand is a fragment of E. coli DNA polymerase I. Incubating the purified first cDNA strand in the presence of the Renow fragment Synthesized (Mania Nice mentioned above). This in vitro synthesized double-stranded cDNA Heavy chain specific of Aspergillus oryzae The ends were made blunt by the action of endonuclease S1 (Mania Nice, supra).
この平滑末端ｃＤＮＡは、１５〜３０％のニュートラル蔗糖勾配によってサイズ 毎に分画した。各両分のサイズ分布は、マガロースゲル電気泳動で既知の［ｌＮ Ａ断片をマーカーにして検定した。約５００ｂｐより長いｃＤＮＡを含む両分を ストックし、ｃＤＮＡをエタノール沈殿によって集めた。This blunt-ended cDNA was sized by a 15-30% neutral sucrose gradient. Each fraction was fractionated. The size distribution of each component is [lN The A fragment was used as a marker for assay. Both parts containing cDNA longer than about 500 bp Stock and cDNA was collected by ethanol precipitation.
例２ ＰＴＨｃＤＮＡのブースミドＢＲ３２２へのクローニングと Ｋ１２ＢＪ、５１ ８３の多 − 約２０塩基長のｄ （Ｃ）テイルをｃＤＮＡの３′末端にターミナルデオキシヌ クレオチジルトランスフェラーゼによって付加した。（マニアナイス前出）この ｄ （Ｃ）テイルが付加されたｃＤＮＡをＰｓｔ Ｉで切断し、ｄ （Ｇ）テイ ルが付加されたベクターｐＢＲ３２２にアニールさせ、作成された組み換えプラ スミドＤＮＡをハナハン（Ｈａｎａｈａｎ、Ｄ）の方法（１６６、Ｊ、Ｍｏ１． Ｂｉｏｌ、 ５５７−５８０１９８３）によって形質転換可能になった大腸菌に １２．Ｂ、Ｊ、５１８３細胞に形質転換した。得られた３３０００の形質転換株 がＰＴＨｃＤＮＡを含んでいることは、コロニーハイブリダイゼーション（Ｈａ ｎａｈａｎ、Ｄ、Ｍｅｓｅｌｓｏｎ １０ Ｇｅｎｅ ６３１９８０）によって 検定した。Example 2 Cloning of PTH cDNA into boothmid BR322 and K12BJ, 51 83 many - Approximately 20 bases long d(C) tail is attached to the 3' end of the cDNA at the terminal deoxynucleotide. Added by cleotidyl transferase. (Mania nice mentioned earlier) This d (C) Cut the cDNA with the tail added with PstI, and d (G) tail the cDNA. The recombinant plate created by annealing the vector pBR322 to which the Sumid DNA was extracted using the method of Hanahan (D) (166, J, Mo1. Biol, 557-5801983). 12. B, J, transformed into 5183 cells. 33,000 transformed strains obtained The fact that Ha contains PTH cDNA was confirmed by colony hybridization (Ha nahan, D., Meselson 10 Gene 631980) Tested.
２〜３４個の形質転換株を、それぞれ８２■直径のニトロセルロースフィルター に直接にブレーティングによって接種し、テトラサイクリンを含む高栄養寒天プ レート上に置き、３７゛ｃで約０．１肺のコロニーが出現する迄インキュベート した。このフィルターに新しい２枚のフィルターを順次押しつけることによって それぞれのフィルターの２枚のレプリカを得た。このレプリカフィルターを新し いテトラサイクリンを含む寒天プレートの上に置き、約直径０．５膿のコロニー が出現するまで３７℃でインキュベートした。細菌のコロニーのマスターフィル ターは寒天プレート上に置き４°Ｃで保存し、２連のレプリカフィルターは寒天 プレートから引きはなして以下のコロニーハイブリダイゼーションの操作に供し た。Transfer 2 to 34 transformants to 82-diameter nitrocellulose filters. inoculated by direct blating onto a highly nutritious agar plate containing tetracycline. Place on plate and incubate at 37°C until approximately 0.1 lung colony appears. did. By sequentially pressing two new filters onto this filter Two replicas of each filter were obtained. Update this replica filter Place colonies approximately 0.5 pus in diameter on agar plates containing tetracycline. The cells were incubated at 37°C until the appearance of . Bacterial colony masterfill The filter was placed on an agar plate and stored at 4°C, and the duplicate filter was placed on an agar plate. Remove from the plate and use for the following colony hybridization procedure. Ta.
例３ えＰＴＨｃＤＮＡ ” ・ コロニーと ５ＳＨＰＴＨ−１０クローン如止肘１ それぞれのコロニーの細胞はその場でアルカリおよび塩化ナトリウム処理により こわされ、各々の細菌のクローンの有するＤＮＡが、露出された。フィルターを トリス緩衝液で中和し８０″Ｃで乾燥させることによってＤＮＡがフィルターに 結合する。ｍ胞の破片は６５°Ｃで、界面活性剤ラウリル硫酸ナトリウム（ＳＤ Ｓ）と塩化ナトリウムで洗浄することにより、はとんどが除去されたが、ＤＮＡ は細菌のコロニーがあった位置のフィルターに結合したままである。これらのフ ィルターを６ｘｓｓｃ液（０，９Ｍ ＮａＣ１゜０．０９Ｍクエン酸ナトリウム ）、１×デンハルト液（０，１ｇ／ｍｉ！フィコル（Ｆｉｃｏｌｌ）、０．１ｇ ／−ポリビニルピロリドン、０．１ｇ／ｍｌ！ウシ血清アルブミン） 、１００ ｇ／ｉｆニシン***ＤＮＡ、０．５％ＳＯ５，０，０５％ピロリン酸ナトリウム に３７°Ｃで２時間浸しており、（ウッズ（Ｗｏｏｄｓ、Ｄ、Ｅ、） ６ Ｆｏ ｃｕｓ Ｎｏ、３．１９８４）ハイブリダイゼーションは、ｉｚｐ標識のＤＮＡ プローブが入っているハイブリダイゼーション液（６Ｘ５ＳＣ，Ｉ Ｘデンハル ト液、２０ｇ＃＋ＮｔＲＮＡ、０．０５％ピロリン酸ナトリウム）中で、４２° Ｃで１８時間かけて行った。（ウッズ前出） ハイブリダイゼーションのプローブとして使ったＤＮＡは、ヘンドリーら（前出 ）らによって報告されたヒトＰＴＨｃＤＮＡの配列から推定された２種類の異な る合成デオキシリボオリゴヌクレオチドのうちの１つである。１番目のフ゛ロー フ゛は、２４マーのオートコドン付近の配列に由来するものでありその配列はＴ ＡＣＴＡＴＧＧＡＣＧＴＴＴＴＣＴＧＴＡＣＣＧＡである。第２のオリゴヌクレ オチドは２４７−であり、終止コドンの３１ヌクレオチド下流に位置する制限酵 素Ｘｂａ Ｔの切断部位をまたぐものであり、その配列はＣＴＣＡＡＧＡＣＧＡ ＧＡＴＣＴＧＴＣＡＣＡＴＣＣである。Example 3 PTH cDNA ”・Colony and 5SHPTH-10 clone 1 Cells from each colony were treated in situ with alkali and sodium chloride. The DNA of each bacterial clone was exposed. filter DNA is transferred to the filter by neutralization with Tris buffer and drying at 80"C. Join. The fragments of m-cells were incubated at 65°C and treated with the surfactant sodium lauryl sulfate (SD). Most of the DNA was removed by washing with S) and sodium chloride, but the DNA remains bound to the filter where the bacterial colony was. These frames Filter 6xssc liquid (0.9M NaCl, 0.09M sodium citrate) ), 1x Denhardt's solution (0.1g/mi! Ficoll, 0.1g /-Polyvinylpyrrolidone, 0.1g/ml! bovine serum albumin), 100 g/if herring sperm DNA, 0.5% SO5, 0.05% sodium pyrophosphate (Woods, D, E,) 6 Fo cus No. 3.1984) Hybridization is performed using izp-labeled DNA. Hybridization solution containing probe (6X5SC, IX Denhal solution, 20 g #+NtRNA, 0.05% sodium pyrophosphate) at 42° It took 18 hours at C. (Woods mentioned above) The DNA used as a hybridization probe was used by Hendry et al. ), two different types deduced from the sequence of human PTH cDNA reported by et al. It is one of the synthetic deoxyribo-oligonucleotides. 1st follow F is derived from the sequence near the 24-mer autocodon, and its sequence is T. ACTATGGACGTTTTCTGTACCGA. Second oligonucleotide Otide is 247-, a restriction enzyme located 31 nucleotides downstream of the stop codon. It straddles the cleavage site of elementary XbaT, and its sequence is CTCAAGACGA GATCTGTCACATCC.
” Ｐ −７−ＡＴＰの３２Ｐをオリゴヌクレオチドの５′末端にポリヌクレオ チドキナーゼの作用で結合させ標識した。（マキサム（Ｍａｘａｍ Ａ、Ｍ、） Ｇ１１ｂｅｒｔ Ｌ ６５． Ｍｅｔｈｏｄｓ Ｅｎｚ ｍｏｌ、 ４９９１ ９８０）ハイブリダイズしたフィルターはオートラジオグラフィーの前に４２℃ で６ＸＳＳＣ１０，０５％ピロリン酸ナトリウム液中で洗浄した。二連のレプリ カフィルターの両方の写しにハイブリダイズしていることで判定してみたところ ６６個のクローンが両方のプローブにポジティブであることがわかった。ｃＤＮ Ａライブラリーがストックされているもとのフィルターからそれらを全て回収し て、−７０°Ｃで永久保存するために増殖させた。これらのうち６つをプラスミ ド調製用とエンドヌクレアーゼによる制限酵素地図の更に詳細な解析をするため に選んで調べたところ、開始コドンとＸｂａ Ｉ部位の近接領域でそれぞれサイ ズが異なってきていることを除いては全てが全く同一のものであった。” 32P of P-7-ATP is attached to the 5' end of the oligonucleotide as a polynucleotide. It was bound and labeled by the action of tidokinase. (Maxam A, M,) G11bert L 65. Methods Enzmol, 4991 980) Hybridized filters were incubated at 42°C before autoradiography. and washed in 6XSSC10.05% sodium pyrophosphate solution. double repli It was determined that it was hybridized to both copies of the filter. Sixty-six clones were found to be positive for both probes. cDN Collect them all from the original filter where the A library is stocked. and grown for permanent storage at -70°C. Plasmid 6 of these For the preparation of endonucleases and for more detailed analysis of restriction enzyme maps using endonucleases. When we selected and investigated the site, we found that the sites were located in the vicinity of the start codon and the Xba I site, respectively. Everything was exactly the same except for the different sizes.
例４ クローン５ＳＨＰＴＨ−１０ ｐＳＳＨＰＴＨ−１０クローンをマサキム・ギルバート法（前出）によるＤＮＡ の配列解析に供した０図６にｐｓｓＨＰＴＨ−１０の完全な構造が示しである。Example 4 Clone 5SHPTH-10 The pSSHPTH-10 clone was converted into DNA using the Masakim-Gilbert method (described above). Figure 6 shows the complete structure of pssHPTH-10.
このクローンは４３２ｂｐ長のＰＴＨｃＤＮＡの配列が、ｐＢＲ３２２のＰｓｔ Ｉ部位に挿入されており５′末端に２７のＧ／Ｃ塩基対か３′末端に１７のＧ／ Ｃ塩基対がある。図４にｐｓｓＨＰＴＨ−１０のｃＤＮＡ挿入部分の完全なりＮ Ａ配列が示しである。開始コドンのちょうど前１−５塩基対の欠失があることを 除いてはヘンドリー（前出）らのシーフェンスを同一であり、使用される開始コ ドンに先立つ、報告されている（ヘンドリーら前出）。開始−終止シグナル（Ａ ＴＧＴＧＡＡＧ）は、（下線部が欠失）、２重の開始シグナル（ＡＴＧＡＴＧ） に変わっていた。This clone has a 432 bp long PTH cDNA sequence that is similar to Pst of pBR322. It is inserted in the I site with 27 G/C base pairs at the 5' end or 17 G/C base pairs at the 3' end. There is a C base pair. Figure 4 shows the complete cDNA insert of pssHPTH-10. Arrangement A is shown. There is a deletion of 1-5 base pairs just before the start codon. Identical to the sea fence of Hendry et al., except that the starting code used is has been reported (Hendry et al., supra). Start-stop signal (A TGTGAAG) is (underlined part deleted), double initiation signal (ATGATG) It had changed to
例５ シャトルベク −Ｌ４の ＨＰＴ）Ｉを酵母で発現させる計画が始まる以前に一般的な酵母発現ベクターの 一連のものが作成された。その１つがｐＬ４であり、以下に述べるように後に使 用してｐＳＳαＬＸ ５− ＨＰＴＩ（を作成した。Example 5 Shuttlebek-L4 Before we started planning to express HPT) I in yeast, we developed a general yeast expression vector. A series of things were created. One of them is pL4, which will be used later as described below. pSSαLX 5-HPTI (was created using
ベッヂ（Ｂｅｇｇｓ Ｊ、Ｄ）、Ｖｏｎ Ｗｅｔｔｓｔｅｉｎ Ｄ、Ｆｒ１ｉｓ Ｊ、Ｋｉｅｌｌａｎｄ −Ｂｒａｎｄｔ Ｍ、５ｔｅｎｄｅｒｕｐ、Ａらによ って作成されたプラスミドｐＪＤＢ２０７“マルチコピー酵母プラスミドベクタ ー”（Ｅｄｓ） Ｍｏ１ｅｃｕｌａｒＧｅｎｅｔｉｃｓ ｉｎ Ｙｅａｓｔ （ １９８１） Ａｌｆｒｅｄ Ｂｅｎｚｏｎ Ｓｙｍｐｏｓｉｕｎ Ｖｏｌ、１６ ３８３−３９０）は、−ＪＧ的な発現ベクターのための基本に選んだ。Beggs J, D, Von Wettstein D, Fr1is J, Kielland-Brandt M, 5tenderup, A et al. Plasmid pJDB207 “multicopy yeast plasmid vector -” (Eds) Mo1ular Genetics in Yeast ( 1981) Alfred Benzon Symposium Vol. 16 383-390) was chosen as the basis for the -JG-like expression vector.
これは、ｐＢＲ３２２に由来するｐＡＴ１５３に酵母の２ミクロンＤＮＡのＥｃ ｏＲＩ断片が挿入されている。また、酵母のＬＥＬ１２遺伝子を持っている。ｐ ＪＤ２０７の酵母ｃｉｒ”細胞中でのコピー数は他のプラスミドに比較してはる かに多く、ａｉｒ″株での非選択培養後でもめずらしく安定である。This results in the addition of yeast 2 micron DNA to pAT153 derived from pBR322. The oRI fragment has been inserted. It also has the yeast LEL12 gene. p The copy number of JD207 in yeast cir' cells is much higher than that of other plasmids. It is unusually stable even after non-selective culture in the air'' strain.
ベレフト（Ｐａｒｅｎｔ Ｓ、Ａ、）ＦｅｎｉＩｌｏｒｅ Ｃ，Ｍ、 Ｂｏｓｔ ｉａｎ Ｋ、Ａ、 ”発現用のベクターシステム、サツカロマイセス・セレビシ アエ（Ｓ。Beleft (Parent S, A,) FeniIlore C, M, Bost ian K, A, “Vector system for expression, Saccharomyces cerevisi Ae (S.
ｃｅｒｅｖｉｓｉａｅ）のＤＮＡシークエンスの分析とクローニング″ １゜ｎ …紅 ８３−１３８ （１９８５） ；エルバー） （Ｅｒｈａｒｔ Ｅ、）Ｈ ｏｌｌｅｎｂｅｒｇ Ｃ，Ｐ、″サッカ０フイセス°セレビシアエ（Ｓａｃｃｈ ａｒｏｌＩｌｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）の２ミクロンＤＮＡ組み換えプ ラスミド上の欠損ＬＥＵ２の存在が高コピー数とキユアリングの原因である。” １５６ Ｊ、Ｂａｃｔｅｒｉａｌ ６２５ ６３５（１９８３） 。cerevisiae) DNA sequence analysis and cloning''1゜n ...Beni 83-138 (1985); Elver) (Erhart E,) H ollenberg C, P, "Sacca 0 Fuises ° cerevisiae" 2 micron DNA recombinant protein of ArolIlyces cerevisiae) The presence of defective LEU2 on the lasmid is responsible for the high copy number and curing. ” 156 J, Bacterial 625 635 (1983).
これらの特徴は選択マーカー遺伝子ＬＥＵ２ （またはＬＥｔ１２ｄと言う、ｄ は欠陥の頭文字）のプロモーターが部分的に弱くなっていることと関連があり、 エルハート（前出）、以下のプラスミド作成において変換されない。These characteristics are expressed by the selectable marker gene LEU2 (or LEt12d, d is associated with a partially weakened promoter of Elhart (supra) is not converted in the following plasmid construction.
ＡＤ旧プロモーターを含む１２６０塩基対のＥｃｏＲＩ −Ａｖａ ＩＩ断片を プラスミドｐＡＤ）１０４０から単離した。ＤＮＡポリメラーゼ■のフレノウフ ラグメントと全ての４つのｄＮＴＰとの反応を完全に行わせた後にＢａｍＨＩリ ンカ−を付加してこの断片をｐＪＤＢ２０７の中にある唯一のＲａｍ）ｌ １部 位にクローン化した。逆方向のプロモーターになっているプラスミドから１０５ ０塩基対のｓｐｈ １断片（ｐＪＤＢ ２０７のｓｐｈ １部位からプロモータ ー断片中のＳｐｈ Ｉ部位に至る）を欠失させ、１つのＢａｎＨＩ部位のみを残 した。このプラスミドをｐＡＬＸｌと称した。The 1260 base pair EcoRI-Ava II fragment containing the AD old promoter was It was isolated from plasmid pAD)1040. Frenouf of DNA polymerase■ After the reaction of the fragment with all four dNTPs is complete, the BamHI reaction is performed. Add a linker and convert this fragment into the only Ram)l copy in pJDB207. cloned into position. 105 from a plasmid with a promoter in the opposite direction 0 base pair sph 1 fragment (promoter from sph 1 site of pJDB 207 - Sph I site in the fragment), leaving only one BanHI site. did. This plasmid was called pALXl.
ｐＡＬＸ ｌ中のβラクタマーゼ遺伝子中のＰｓｔ１部位を次に遺伝子を不活性 化させることなく欠失させた。ｐＡＬＸ １をＰｓｔｌで完全に分解し、ヌクレ アーゼＳ１でＰｓｔ１部位を分解し、次にＰｖｕ １とＢｇｌ Ｉによって部分 的に分解した。同時にｐＵｃｌＢがらビエイラ（Ｖｉｅｉｒａ Ｊ、） Ｍｅｓ ｓｉｎｇ Ｊ、 １９． Ｇｅｎｅ、 ２５９．１９８２） Ｐｓｔ １部位を 含まない対応するβラクタマーゼ遺伝子の断片になる。Ｐｖｕ Ｉ ｒＢｇｌ Ｉの２５０塩基対の断片を単離した。これを部分分解したｐＡＬｘｌに結合させ た。単離されたアンピシリン耐性クローン全てのうち、βラクタマーゼ遺伝子は ｐｌＪｃ８断片と結合することにょっ特表千２−５０１１０８ （７） で、復帰していた。このプラスミドのことをｐＡＬＸ ２と称する。The Pst1 site in the β-lactamase gene in pALX is then inactivated. It was deleted without changing. Completely degrade pALX1 with Pstl and remove the nucleic acid. The Pst1 site is degraded with Ase S1, and then the Pst1 site is degraded with Pvu1 and BglI. It was disassembled. At the same time, pUclB was removed from Vieira (Vieira J,) Mes sing J, 19. Gene, 259.1982) Pst 1 site It becomes a fragment of the corresponding β-lactamase gene that does not contain it. Pvu I rBgl A 250 base pair fragment of I was isolated. Combine this with partially degraded pALxl Ta. Among all ampicillin-resistant clones isolated, the β-lactamase gene Special table 12-501108 (7) that binds to the plJc8 fragment So, he was back. This plasmid is called pALX2.
ベルゲン（Ｂｅｒｇｅｎ ）大学のクレッペ（Ｋ、Ｋｌｅｐｐｅ）教授より以下 のオリゴヌクレオチドを購入し、ｐＡＬＸ ２のＢａｗｌ １部位に挿入した。The following from Professor Kleppe of the University of Bergen: The oligonucleotide was purchased and inserted into the Bawl 1 site of pALX2.
適正な方向にプロモーターが入ったプラスミドを単離し、これをｐＡＬＸ ３と 称する。Isolate a plasmid with the promoter in the correct orientation and call it pALX3. to be called.
最終的にｐＡＬＸ ３は旧ｎｄＩ［［で切断し再結合させることによってＨｉｎ ｄ Ｉｎの４８０塩基対断片を欠失させた。このプラスミドをｐＡＬＸ ４と名 づけた。図７にｐＡＬＸ４の制限地図を示しである。Finally, pALX3 was cleaved with old ndI[[ and recombined with Hin. A 480 base pair fragment of dIn was deleted. This plasmid was named pALX4. I attached it. Figure 7 shows the restriction map of pALX4.
ｐＬ４はｐＡＬＸ ４由来のものでＡＤ）ＩＩのプロモーターが欠失している。pL4 is derived from pALX4, and the AD) II promoter is deleted.
ｐＬ４は他のプロモーターの挿入用のものとして使われた。pL4 was used for insertion of other promoters.
ｐＡＬＸ ４をまずＢｇｌ ＩＩと５ａｉｌで切断した。生じてきた接着末端を ＤＮＡポリメラーゼＩのフレノウフラグメントと４つのｄＮＴＰによってうずめ て、再結合させた。この処理によってＡＤＨＩプロモーターが除去され、Ｂｇｌ ｍ部位を再び持つベクターｐＬ４が作成された。pALX4 was first cut with BglII and 5ail. The adhesive ends that have formed Uzume by the Flenow fragment of DNA polymerase I and four dNTPs and recombined it. This treatment removes the ADHI promoter and Vector pL4, which has the m site again, was created.
例６ 出国■ 酵母の性フェロモン９Ｆαｌの遺伝子は初めてカージャン（Ｋｕｒｊａｎ、Ｊ、 ）とへルスコウィッ（Ｈｅｒｓｋｏｗｉｔｚ、 １．）によってクローニングさ れた。Example 6 Departure■ The gene for the yeast sex pheromone 9Fαl was first discovered by Kurjan (J. ) and Herskowitz (1.). It was.
°゛酵母フェロモン遺伝子（ＭＦα）の構造；推定上のα因子前駆体には４つの タンデムコピーの成熟の因子が含まれる”３０並旦、９３３−９４３ （１９８ ２）。この文献に報告されている配列をもとに肝α１遺伝子を再クローニングし た。全酵母ＤＮＡをＹ２８８Ｃ株より得、ＥｃｏＲＩで切断し、この切断産物の うち１．６〜１．８ｋｄの範囲のサイズのものを調整用アガロースゲルから単離 した。これは次に脱リン酸化され、ＥｃｏＲＩで切断されたｐＢＲ３２２に結合 させ、大腸菌ＢＪ５１８３を形質転換するのに使われた。生じてきたクローンを 下記の構成の標識オリゴヌクレオチドでハイブリダイゼーションによって）ＩＦ α１遺伝子の挿入を調べた。°゛Structure of the yeast pheromone gene (MFα); the putative α-factor precursor contains four 30 Nandan, 933-943 (198 2). The liver α1 gene was recloned based on the sequence reported in this literature. Ta. Total yeast DNA was obtained from Y288C strain, cut with EcoRI, and the cut product was Of these, those in the size range of 1.6 to 1.8 kd were isolated from agarose gel for preparation. did. This is then dephosphorylated and binds to EcoRI-cleaved pBR322. and used to transform E. coli BJ5183. The clone that has arisen IF by hybridization with labeled oligonucleotides of the following configuration: Insertion of the α1 gene was investigated.
ＴＧＧＣＡＴＴＧＧＣＴＧＣＡＡＣＴＡＡＡＧＣポジティブクローンより精製し てきたＤＮＡは、次に、プラスミドＤＮＡが調製されてきた大腸菌ＪＡ２２１を 形質転換するのに使われた。以下のプラスミドに使われたプラスミドすなわちｐ ＭＦα１−１は図８に示しである。Purified from TGGCATTGGCTGCAAACTAAAGC positive clone. The resulting DNA is then injected into Escherichia coli JA221, from which plasmid DNA has been prepared. used for transformation. The plasmids used for the following plasmids i.e. p MFα1-1 is shown in FIG.
ＰＭＦα−１はＥｅｏＲＩで切断し、ＤＮＡポリメラーゼＩのフレノウフラグメ ントと４つのｄＮＴＰと反応させ、フェノール抽出を行いＢｇｌ Ｉｆで切断し た。１．７ｋｂの肝α１遺伝子をアガロースゲルから単離した。酵母シャトルベ クターにこれを挿入する前に、ｐＬ４の旧ｎｄＩ１１部位をＨｉｎｄＩＩＩによ る分解によって除去し、フレノウフラグメントでのうめあわせ反応と、再結合に よってｐＬ５シャトルベクターを作成した。ｐＬ５はＢａＩＩＨＩが切断され、 ＤＮＡポリメラーゼＩのフレノウフラグメントと４つのｄＮＴＤによってうめあ わせ、フェノール抽出をし、Ｂｇｌ ＩＩで切断した。ゲルで精製した後にこれ をＭＦα１の断片と結合させ、図８に示しであるような発現ベクターｐαＬＸ５ を作成した。PMFα-1 was cleaved with EeoRI and injected with the Flenow fragment of DNA polymerase I. react with four dNTPs, perform phenol extraction, and cleave with BglIf. Ta. The 1.7 kb liver α1 gene was isolated from an agarose gel. yeast shuttlebe Before inserting it into the vector, the old ndI11 site of pL4 was removed with HindIII. removed by decomposition, followed by filling reaction with Flenow fragments and recombination. Therefore, a pL5 shuttle vector was created. pL5 is cleaved with BaIIHI, Umea by the Flenow fragment of DNA polymerase I and four dNTDs It was washed, extracted with phenol, and cleaved with Bgl II. This after gel purification was combined with a fragment of MFα1 to create the expression vector pαLX5 as shown in FIG. It was created.
例７ ＳＳｃｒＬＸ５−ＨＰＴＨ１（７） ｊ＠ｐｓｓＨＰＴＨ−１０プラスミドから の２８８塩基対のＢｇｌ Ｉｔ、Ｘｂａ Ｉ断片を単離しＰＯＣｌ２にそのベク ターのＢａｍＨＩとＸｂａ Ｉ部位を用いてサブクローニングした。このｐｔｌ ｃ−）ＩＰＴＦＩと名づけられたサブクローンをＤｐｎ Ｉで切断し最大の断片 を単離した。この断片をＳａｌ ｌで切断し、Ｓａｌ ｌ切断の方は接着末端に Ｄｐｎ ｌ切断の方は平滑末端になっている、２つの生成断片のうち小さい方を 単離した。Example 7 SScrLX5-HPTH1 (7) from j@pssHPTH-10 plasmid A 288 base pair Bgl It, Xba I fragment was isolated and inserted into POCl2 as a vector. Subcloning was performed using the BamHI and XbaI sites of the target. This ptl c-) Cut the subclone named IPTFI with DpnI to obtain the largest fragment. was isolated. Cut this fragment with Sal 1, and the Sal 1 cut ends with sticky ends. The Dpnl cleavage has a blunt end, and the smaller of the two generated fragments is isolated.
ｐαＬＸ５を旧ｎｄＩＩｌで切断し、ＤＮＡポリメラーゼ■のフレノウフラグメ ントと４つのｄＮＴＰでのうめあわせ反応をし、フェノール抽出を行い、Ｓａｌ １での切断を行った。ゲルから精製後、これを上述のＨＰＴ）Ｉ断片に結合し た。このり°ローンはリーディングフレームが適正となるように旧ｎｄｌＩ［部 位が再生されている。pαLX5 was cut with old ndIIl, and the Flenow fragment of DNA polymerase The sample was reacted with four dNTPs, extracted with phenol, and extracted with Sal. The cutting at step 1 was performed. After purification from the gel, this was ligated to the HPT)I fragment described above. Ta. This loan was created using the old ndlI[part] so that the reading frame was correct. is being played.
このプラスミドをｐＳＳαＬＸ ５−ＨＰＴＨ１と称し、図９に示しである。肝 α１−ＨＰＴＨ融合遺伝子の配列は図５に示しである。This plasmid was designated pSSαLX5-HPTH1 and is shown in FIG. liver The sequence of the α1-HPTH fusion gene is shown in FIG.
例８ − にお番るＨＰＴＨの とゝパ・ 酵母株ＰＬ２００（ｃｒ、 Ｕｒａ３． Ｌｅｕ２）を、プラスミドｐαＬＸ５ とｐＳＳαＬＸ ５− ＨＰＴ）Ｉ ｌで、スフェロプラスト法で形質転換させ た。Example 8 - HPTH's and Pa. Yeast strain PL200 (cr, Ura3.Leu2) was transformed into plasmid pαLX5 and pSSαLX 5-HPT) I by the spheroplast method. Ta.
それぞれのうち１つの形質転換株をＬｅｕ−の培地で成育させ、細胞を含まない 培養液を５ＯＳ−ＰＡＧＥ銀染色で調べた。（図１２）ｐＳＳαＬＸ ５− Ｈ ＰＴＨ１の形質転換株の培地には２つの大量のバンドがあったが、ｐαＬＸ５の 形質転換株培養液にはなかった。１分子量に対応し、他のバンドは約１６０００ ダルトンであり、プロセンシングを受けていない肝α１−）ＩＰＴＨ融合産物に 対応するものであろう。両ポリペプチド共に生来のホルモンと同一の態様で抗ヒ ） ＰＴＨ抗体と反応した。One transformant of each was grown in Leu medium, free of cells. The culture fluid was examined by 5OS-PAGE silver staining. (Figure 12) pSSαLX 5-H There were two large bands in the medium of the PTH1 transformant, but there were two large bands in the medium of the PTH1 transformant, but It was not present in the culture solution of the transformed strain. 1 molecular weight, the other bands are approximately 16,000 dalton and unprocessed liver α1-)IPTH fusion product. It will correspond. Both polypeptides act as antihuman agents in the same manner as natural hormones. ) Reacted with PTH antibody.
以上の例は、例証により示されているが、本発明はこれに制限されるわけではな い。以上の例はヒト副甲状腺ホルモンを生産、分泌するサツカロマイセス・セレ ビシアエ（Ｓ、ｃｅｒｅｖｉｓｉａｅ）の開発を目的としたものだが、本発明の 方法は、どの種の副甲状腺ホルモンをコードするＤＮＡを含むプラスミドを作成 するのにも応用され得よう。更にこのプラスミドはどの種の酵母にも挿入され得 よう。故に本発明はサツカロマイセス・セレビシアエ（Ｓ、ｃｅｒｅｖｉｓｉａ ｅ）に制限されない。Although the above examples are given by way of illustration, the invention is not limited thereto. stomach. The above example is Satucharomyces cere, which produces and secretes human parathyroid hormone. The purpose of this invention is to develop S. cerevisiae. The method involves creating a plasmid containing DNA encoding any species of parathyroid hormone. It could also be applied to Furthermore, this plasmid can be inserted into any species of yeast. Good morning. Therefore, the present invention relates to S. cerevisiae (S. cerevisiae). e) is not limited to.
本発明の酵母が生産した、クローン化ヒト副甲状腺ホルモンには既知の、又は潜 在的可能性のある、様々な使用法がある。The cloned human parathyroid hormone produced by the yeast of the present invention has known or latent There are various possible uses.
例えば、最近の医学理論によると、ヒト副甲状腺ホルモンは骨粗ｒ症の治療に非 常に効果的であるらしい。遺伝子工学的に作成された副甲状腺ホルモンはヒトや 動物の副甲状腺ホルモンレベルを測る分析キットについても利用価値があろう。For example, recent medical theory suggests that human parathyroid hormone has no role in the treatment of osteoporosis. It always seems to be effective. Genetically engineered parathyroid hormone can be used in humans and Analytical kits for measuring parathyroid hormone levels in animals may also be of value.
ヒト副甲状腺ホルモンやその断片は減少性又は病理的血中カルシウムレベル異常 を示すヒトや動物の治療にも使えるであろう。Human parathyroid hormone or its fragments are associated with decreased or pathological blood calcium level abnormalities. It could also be used in the treatment of humans and animals exhibiting this disease.
遺伝子工学的に作られた副甲状腺ホルモンが例えば本発明の結果として大量に入 手可能になると、数多くの他の使用法の開発が期待される。For example, genetically engineered parathyroid hormone may be available in large quantities as a result of the present invention. Once available, numerous other uses are expected to be developed.
本発明は特定の好適具体例を参照しつつ記載したが、その応用は当業者に明白な ので、本発明は上記具体例に限定されない。Although the invention has been described with reference to certain preferred embodiments, its applications will be apparent to those skilled in the art. Therefore, the present invention is not limited to the above specific examples.
人ＴＧ八へＨＣＣＮＧＣＮＡＡＲＧＡＹ入τにＧ（ＮＡＡＲＧＴＮＡτＧλＴＨ Ｇ）ＮＡＴＧＹτＮＧＣＮ入ＴｇτＧＹτＴＹＹτＮ７０ ＣＯ１１０ ＡＣＮλλＲＷＳＮＧ、ＡＹＧＧＮＡＡＲＷＳＮＧＴＮＡＡＲＡ入Ｒ１’、ＧＮ ＷＳＮＧτ９ＷＳＮＧ入ＲＡτＨ（λＲＹτＮＡＴＧＣＡＹ人ＡＹＹＴＮＧＧＮ 入ＡＲＣＡＹＹＴＮ入入Ｙ贅ＳＮ入τにＧＡＲＭＧＮＧ）ＮＧλＲＴＧＧＹτＮ ＭＧＮ入λＨＡλＲＹＴＮＣ入ＲＧＡＹＧＴＮＣ入ＹλλＹττ”Ｇ’：’ＮＧ ＣＮＹＴＮＧＧＮＧ（ＮＣＣＮＹＴＮＧＣＮＣＣＮＭＧＮＧλＹＧＣＮＧＧＮ讐 ＳＮＣ入Ｒ２５０’ ２７０ ２９０ ＭＧＮＣＣＮＭＧＮＡＡＲＡＡＲＧＡＲＧλＹ入入ＹＧＴＮＹτＮＧτＮＧＡＲ ＷＳＮＣＡＹＧＡＲ入ＡＲＷＳＮＹτＮＧＧＮＧλＲコ１０ ３コ０ ＧＣＮＧＡ”ＡＡＲＧＣＮＧＡＹＧＴＮＡＡＹＧＴＮＹＴＮＡＣＮ入ＡＲＧＣＮ 入ＡＲＷＳＮＣλＲＴ！’ＩＲＨ雪入ｏ【Ｃ Ｒ露 Ａ ｏｒ Ｇ ｗ諺入口【Ｔ Ｓ冨Ｃｏｒ Ｇ Ｙ ｍ Ｃｏｒで Ｈｍ Ａ ｏｒ Ｃｏ： Ｔ Ｎ 冨 Ａ ｏｒ Ｇ ｏｒ Ｃｏｒ Ｔ。G(NAARGTNAτGλTH G) NATGYτNGCN entered TgτGYτTYYτN70 CO110 ACNλλRWSNG, AYGGNAARWSNGTNAARA entered R1', GN WSNGτ9WSNG enter RAτH (λRYτNATGCAY person AYYTNGGN Enter ARCAYYTN Enter Y SN enter τ に GARMGNG) NGλRTGGYτN MGN input λHAλRYTNC input RGAYGTNC input YλλYττ”G’:’NG CNYTNGGNG(NCCNYTNGCNCCNMGNGλYGCNGGN) R250' 270 290 with SNC MGNCCNMGNAARAARGARGλYENTRYGTNYτNGτNGAR WSNCAYGAR entered ARWSNYτNGGNGλRko10 3ko0 GCNGA"AARGCNGAYGTNAAYGTNYTNACNARGCN Enter ARWSNCλRT! 'IRH Yukiiri o [C R dew A or G W proverb entrance [T S Tomi Cor G At Ym Cor Hm A or Co: T N Tomi A or G or Cor T.
Ｆ工ＧＬＩＲＥ １ 人ＴＣλＴＧＡＴＡＣＣＴＧＣＡＡＡＡＧＡＣＡＴＧＧＣＴＡＡＡＧＴＴＡＴＧ ＡＴＴＧＴＣＡＴＧττＧＧＣ入ＡＴＴＴＧＴＴＴＴＣＴＴＡＣＡＡＡＡＴＣＧ ＧＡＴＧＧＧＡＡＡＴＣＴＧＴＴＡＡＧＡＡＧＡＧＡＴＣＴＧＴＧＡＧＴＧＡＡ ＡＴＡＣＡＧＣＴ”ＡＴＧＣＡＧＡＧＧＣＣＣＣＧ入ＡＡＡＡＡＧＧ入λＧＡＧ λ入ＴＧＴＣττＧＧＴＴＧＡＧＡＧＣＣＡＴＧＡλ人入λＡＧＴＣＴτＧＧλ Ｆ工ＧＵＰ、Ｅ ２ ＡＣＡＧＡＴ入ττＧＴＣλＧ入ＧＴＴＣτＧＣＴＣτ入ＧＡ（ＡＧＴＧＴＡＧ ＧＧＣＡＡＣλＡτＡＣλＴＣＣτＧＣτＡＡττＣＡＡＡＧＣＴＣτλττ入 Ｍ−人ｏｒ Ｃ Ｒ雪Ａ ０＋” Ｇ 誓璽Ａｏｒで Ｓ Ｗ ＣＯｒ ？ Ｙ冨Ｃｏ【で Ｈ−人Ｏｒ （Ｏｒ Ｔ ・Ｎ ｍ Ａ ｏｒ Ｇ ｏｒ Ｃｏｒ ？。F Engineering GLIRE 1 PersonTCλTGATAACCTGCAAAAGACATGGCTAAAGTTATG ATTGTCATGττGGC ATTTGTTTTCTTACAAAATCG GATGGGAAATCTGTTAAGAAGAGAGATCTGTGAGTGAA ATACAGCT”ATGCAGAGGCCCCG into AAAAAAGG into λGAG λ entering TGTCττGGTTGAGAGCCATGAλ entering λAGTCTτGGλ F Engineering GUP, E2 ACAGAT input ττGTCλG input GTTCτGCTCτ input GA (AGTGTAG GGCAACλAτACλTCCτGCτAAττCAAGCTCτλττ input M-person or C R snow A 0+”G With the oath Aor SW COr? Y Tomi Co H-Person Or (Or T ・Nm A or G or Cor? .
Ｆ工ＧＵＲＥ ３ ＡＡＡＧＣＴＣτ入τＴＡ。F Engineering GURE 3 AAAGCTCτinτTA.
ＦＩＧＵＲＥ ａ １０ コ０ ５０ ＡＣＡＧ八τＡＴＴＧＴＣＡＧλＧＴＴＣＴＧＣ’ｒＣＴＡＧＡ（ＡＧＴＧτλ ＧＧＧＣ入ＡＣＡＡＴＡＣλＴＧＣＴＧ（’ｒ入ＡττＣ゛入λＡＧＣＴＣＴＡ ττＡ。FIGURE a 10 ko 0 50 ACAG8τATTGTCAGλGTTCTGC’rCTAGA(AGTGτλ GGGC enters ACAATACλTGCTG('r enters AττC゛enters λAGCTCTA ττA.
罷３ハ ＦＩＧＵＲＥ ６ ＦＩＧＵＲＥ ７ Ｆ工ＧＵＲＥ １０ 人ＲＧ入ＲＧＡＹλＡＹＧＴＮＹフＮＧτＮＧ入ＲＷＳＮＣＡＭＧλＲＡＡＲＷ ＳＮＹτＮＧＧＮＧＡＲＧＣＮＧＡＹ入ＡＦＩＧＣＮＧＴτＴ？ＣＡＴＴ’：” ＣＴＣＣＧＴＡＡＡ（入λＣＣＴＧ’：″：Ｔ”：ＣＣＣλτＧでＡＡＴ入ＴＣ ＣτＴττＣＴＡＴＴＴＴＴＣＧＴsＴ ＣＧττＡＣＣＡＡＣＴττλＣ入ＣＡＴＡＣＴＴ’：入τＡＴＡＧＣＴＡ？、 讐ｈｅｒｅｉｎＦ工ＧＵＲＥ １０ （Ｃｏｎｔ、） Ｆ工ＧＵＲＥ １１ ττＴＴＣＡＴＴフＣτＣＣＧフＡＡＡＣＡＡＣＣτＧＴＴＴＴＣＣＣＡＴＧτ 入ＡＴλτＣＣττττＣτ入ττＴττＣＧτττＦＩＧＵＲ三 １１ （Ｃ ｏｎ；、） 国際調査報告 ｌｍａｍｌｍ−＾−−に一−Ｉｅ、ｐｃ＝７＝ｐ ８７１００５８１国際調査報 告 ＥＰ ８７００５８１ ＳＡ １９１０２Suspension 3c FIGURE 6 FIGURE 7 F Engineering GURE 10 Human RG entered RGAYλAYGTNYFNGτNG entered RWSNCAMGλRAARW SNYτNGGNGARGCNGAY entered AFIGCNGTτT? CATT’:” CTCCGTAAA (input λCCTG’:″:T”: AAT input TC with CCCλτG CτTττCTATTTTTCGTsT CGττACCAACTττλC input CATACTT’: input τATAGCTA? , Herein F Engineering GURE 10 (Cont,) F Engineering GURE 11 ττTTCATTFCτCCGFAAACAAACCτGTTTTCCCATGτ Input ATλτCCττττCτInput ττTττCGτττFIGUR3 11 (C on;,) international search report lmamlm-^--ni-Ie, pc=7=p 87100581 International Research Report Notice EP 8700581 SA 19102
Claims (29)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US92168486A | 1986-10-22 | 1986-10-22 | |
| US921,684 | 1986-10-22 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9082921A Division JP2834111B2 (en) | 1986-10-22 | 1997-04-01 | Microbial production of human parathyroid hormone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02501108A true JPH02501108A (en) | 1990-04-19 |
| JP2777132B2 JP2777132B2 (en) | 1998-07-16 |
Family
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Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62506628A Expired - Lifetime JP2777132B2 (en) | 1986-10-22 | 1987-10-07 | Microbial production of human parathyroid hormone |
| JP8179694A Expired - Lifetime JP2828427B2 (en) | 1986-10-22 | 1996-07-09 | Microbial production of human parathyroid hormone |
| JP9082921A Expired - Lifetime JP2834111B2 (en) | 1986-10-22 | 1997-04-01 | Microbial production of human parathyroid hormone |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8179694A Expired - Lifetime JP2828427B2 (en) | 1986-10-22 | 1996-07-09 | Microbial production of human parathyroid hormone |
| JP9082921A Expired - Lifetime JP2834111B2 (en) | 1986-10-22 | 1997-04-01 | Microbial production of human parathyroid hormone |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US6962796B1 (en) |
| EP (3) | EP0604706A1 (en) |
| JP (3) | JP2777132B2 (en) |
| AU (2) | AU627147B2 (en) |
| DE (2) | DE3752375T8 (en) |
| HK (1) | HK68497A (en) |
| WO (1) | WO1988003165A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010523093A (en) * | 2007-04-05 | 2010-07-15 | シンバイオテック ゲゼルシャフト ツーア フォルシュング ウント エントヴィックルング アウフ デム ゲビート デア ビオテヒノロギー ミット ベシュレンクテル ハフツング | Bis-met histone |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE37919E1 (en) | 1989-05-12 | 2002-12-03 | The General Hospital Corporation | Recombinant DNA method for production of parathyroid hormone |
| US5171670A (en) * | 1989-05-12 | 1992-12-15 | The General Hospital Corporation | Recombinant dna method for production of parathyroid hormone |
| WO1991005050A1 (en) * | 1989-09-29 | 1991-04-18 | Her Majesty In Right Of Canada As Represented By The National Research Council Of Canada | Synthesis of mature human parathyroid hormone |
| EP0483509B1 (en) * | 1990-09-28 | 1995-07-19 | Takeda Chemical Industries, Ltd. | Synthetic gene coding for human parathyroid hormone |
| JPH05271279A (en) * | 1991-08-07 | 1993-10-19 | Takeda Chem Ind Ltd | Human parathyroid hormone mutein and its production |
| ATE152127T1 (en) * | 1991-08-07 | 1997-05-15 | Takeda Chemical Industries Ltd | MUTEINS OF THE HUMAN PARATHORMONE AND THEIR PRODUCTION |
| US5382658A (en) * | 1992-04-03 | 1995-01-17 | Allelix Biopharmaceuticals Inc. | Stability-enhanced variants of parathyroid hormone |
| CA2098639A1 (en) * | 1992-06-19 | 1993-12-20 | K. Anne Kronis | Bone stimulating, non-vasoactive parathyroid hormone variants |
| US5830853A (en) | 1994-06-23 | 1998-11-03 | Astra Aktiebolag | Systemic administration of a therapeutic preparation |
| US6632456B1 (en) | 1993-06-24 | 2003-10-14 | Astrazeneca Ab | Compositions for inhalation |
| US6794357B1 (en) | 1993-06-24 | 2004-09-21 | Astrazeneca Ab | Compositions for inhalation |
| US6524557B1 (en) | 1994-12-22 | 2003-02-25 | Astrazeneca Ab | Aerosol formulations of peptides and proteins |
| KR100386836B1 (en) * | 2000-08-31 | 2003-06-09 | 동국제약 주식회사 | Transformant of Yeast Producing Recombinant human parathyroid hormone and Method for Producing Said hormone |
| EP1985628A1 (en) * | 2007-04-05 | 2008-10-29 | SYMBIOTEC Gesellschaft zur Forschung und Entwicklung auf dem Gebiet der Biotechnologie mbH | Bis-met histones |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6034986A (en) * | 1983-04-11 | 1985-02-22 | ゲゼルシヤフト・フユア・ビオテクノロギツシエ・フオルシユンク・ミツト・ベシユレンクテル・ハフツング | Hybrid vector for producing human paratolumone and human paratolumone gene |
| US4546082A (en) * | 1982-06-17 | 1985-10-08 | Regents Of The Univ. Of California | E. coli/Saccharomyces cerevisiae plasmid cloning vector containing the alpha-factor gene for secretion and processing of hybrid proteins |
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| US3886132A (en) | 1972-12-21 | 1975-05-27 | Us Health | Human parathyroid hormone |
| US4468464A (en) | 1974-11-04 | 1984-08-28 | The Board Of Trustees Of The Leland Stanford Junior University | Biologically functional molecular chimeras |
| US4237224A (en) | 1974-11-04 | 1980-12-02 | Board Of Trustees Of The Leland Stanford Jr. University | Process for producing biologically functional molecular chimeras |
| US4264731A (en) | 1977-05-27 | 1981-04-28 | The Regents Of The University Of California | DNA Joining method |
| US4366246A (en) | 1977-11-08 | 1982-12-28 | Genentech, Inc. | Method for microbial polypeptide expression |
| US4425437A (en) | 1979-11-05 | 1984-01-10 | Genentech, Inc. | Microbial polypeptide expression vehicle |
| US4338397A (en) | 1980-04-11 | 1982-07-06 | President And Fellows Of Harvard College | Mature protein synthesis |
| US4383066A (en) | 1980-04-15 | 1983-05-10 | Mitsubishi Gas Chemical Company, Inc. | Polyphenylene ether resin composition |
| JPS5825439B2 (en) * | 1980-12-30 | 1983-05-27 | 株式会社林原生物化学研究所 | Method for producing human parathyroid hormone |
| US4394443A (en) | 1980-12-18 | 1983-07-19 | Yale University | Method for cloning genes |
| US4624926A (en) | 1981-01-02 | 1986-11-25 | The Research Foundation Of State University Of New York | Novel cloning vehicles for polypeptide expression in microbial hosts |
| NO163060C (en) | 1981-03-13 | 1990-03-28 | Damon Biotech Inc | PROCEDURE FOR THE PREPARATION OF SUBSTANCES MADE BY LIVING CELLS PRODUCING INTERFERONES OR ANTIBODIES. |
| US4532207A (en) | 1982-03-19 | 1985-07-30 | G. D. Searle & Co. | Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase |
| WO1984001173A1 (en) * | 1982-09-15 | 1984-03-29 | Immuno Nuclear Corp | Production of mature proteins in transformed yeast |
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| US5420242A (en) * | 1986-10-22 | 1995-05-30 | Kaare M. Gautvik | Production of human parathyroid hormone from microorganisms |
| USRE37919E1 (en) * | 1989-05-12 | 2002-12-03 | The General Hospital Corporation | Recombinant DNA method for production of parathyroid hormone |
| US5171670A (en) | 1989-05-12 | 1992-12-15 | The General Hospital Corporation | Recombinant dna method for production of parathyroid hormone |
-
1987
- 1987-10-07 AU AU81048/87A patent/AU627147B2/en not_active Expired - Fee Related
- 1987-10-07 DE DE3752375T patent/DE3752375T8/en active Active
- 1987-10-07 EP EP93112644A patent/EP0604706A1/en not_active Withdrawn
- 1987-10-07 EP EP01107746A patent/EP1114865B1/en not_active Expired - Lifetime
- 1987-10-07 DE DE3789316T patent/DE3789316T2/en not_active Expired - Lifetime
- 1987-10-07 WO PCT/EP1987/000581 patent/WO1988003165A1/en active IP Right Grant
- 1987-10-07 EP EP87906915A patent/EP0383751B1/en not_active Expired - Lifetime
- 1987-10-07 JP JP62506628A patent/JP2777132B2/en not_active Expired - Lifetime
-
1992
- 1992-11-20 AU AU28516/92A patent/AU665034B2/en not_active Expired
-
1996
- 1996-07-09 JP JP8179694A patent/JP2828427B2/en not_active Expired - Lifetime
-
1997
- 1997-04-01 JP JP9082921A patent/JP2834111B2/en not_active Expired - Lifetime
- 1997-05-22 HK HK68497A patent/HK68497A/en not_active IP Right Cessation
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- 2000-09-25 US US09/668,154 patent/US6962796B1/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4546082A (en) * | 1982-06-17 | 1985-10-08 | Regents Of The Univ. Of California | E. coli/Saccharomyces cerevisiae plasmid cloning vector containing the alpha-factor gene for secretion and processing of hybrid proteins |
| JPS6034986A (en) * | 1983-04-11 | 1985-02-22 | ゲゼルシヤフト・フユア・ビオテクノロギツシエ・フオルシユンク・ミツト・ベシユレンクテル・ハフツング | Hybrid vector for producing human paratolumone and human paratolumone gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010523093A (en) * | 2007-04-05 | 2010-07-15 | シンバイオテック ゲゼルシャフト ツーア フォルシュング ウント エントヴィックルング アウフ デム ゲビート デア ビオテヒノロギー ミット ベシュレンクテル ハフツング | Bis-met histone |
| JP2014209909A (en) * | 2007-04-05 | 2014-11-13 | シンバイオテック ゲゼルシャフト ツーアフォルシュング ウント エントヴィックルング アウフ デム ゲビート デア ビオテヒノロギー ミット ベシュレンクテル ハフツング | Bis-met histones |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH1014587A (en) | 1998-01-20 |
| DE3752375T8 (en) | 2005-03-10 |
| EP1114865B1 (en) | 2004-01-21 |
| JP2777132B2 (en) | 1998-07-16 |
| AU2851692A (en) | 1993-04-08 |
| AU665034B2 (en) | 1995-12-14 |
| JPH09110899A (en) | 1997-04-28 |
| EP0383751A1 (en) | 1990-08-29 |
| DE3789316T2 (en) | 1994-08-25 |
| EP0604706A1 (en) | 1994-07-06 |
| JP2828427B2 (en) | 1998-11-25 |
| US6962796B1 (en) | 2005-11-08 |
| JP2834111B2 (en) | 1998-12-09 |
| DE3752375T2 (en) | 2004-10-21 |
| HK68497A (en) | 1997-05-30 |
| WO1988003165A1 (en) | 1988-05-05 |
| DE3789316D1 (en) | 1994-04-14 |
| AU8104887A (en) | 1988-05-25 |
| AU627147B2 (en) | 1992-08-20 |
| DE3752375D1 (en) | 2004-02-26 |
| EP0383751B1 (en) | 1994-03-09 |
| EP1114865A1 (en) | 2001-07-11 |
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